The synthesis and manufacture of L-ascorbic acid (AsA, vitamin C) has received considerable attention due to its relatively large market volume and high value as a vitamin and antioxidant. A chemical route, the Reichstein-Grussner method, from glucose to AsA, was first disclosed in 1934 (Helv. Chim. Acta 17:311-328). More recently bioconversion methods for the production of AsA intermediates have been disclosed and reference is made to Lazarus et al. (1989), Vitamin C: Bioconversion via a Recombinant DNA Approach”, GENETICS AND MOLECULAR BIOLOGY OF INDUSTRIAL MICROORGANISMS, American Society for Microbiology, Washington D.C. edited by C. L. Hershberger; Crawford et al., (1980) Advances in Carbohydrate Chemistry and Biochemistry 37: 79-155 (1980); Anderson et al., (1985) Sci. 230: 144-149; and Sugisawa et al. (1990) Agric. Biol. Chem. 54:1201-1209.
A typical process for the manufacture of AsA is shown in FIG. 15. In general, the process begins with a metabolite used by a microorganism, e.g. D-glucose. Through enzymatic conversions, which may include D-glucose dehydrogenase, D-gluconate dehydrogenase, and 2-keto-D-gluconate dehydrogenase, the D-glucose undergoes a series of oxidative steps to yield 2,5-diketo-D-gluconate (2,5-DKG). Further the 2,5-DKG may be reduced to 2-keto-L-gulonic acid (2-KLG). This process may be carried out in microorganisms such as Gluconobacter, Acetobacter, Erwinia or Pantoea. Reference is made to various U.S. patents disclosing parts of this overall conversion such as U.S. Pat. Nos. 3,790,444; 3,922,194; 3,959,076; 3,998,697; 4,245,049 and 5,008,193. Because of the commercial market for AsA, AsA intermediates independent of AsA, have become a material of economic and industrial importance and for that reason it would be desirable to increase microbial efficiency for enzymatic conversion of carbon substrates into AsA intermediates.
Compared to other bacterial organisms, the gram-negative Enterobacteria, Pantoea citrea has the ability to efficiently convert glucose and other sugars into different aldo and keto-sugar derivatives and particularly into the AsA intermediates 2,5-DKG and 2-KGL. In this invention, the genome of Pantoea citrea was analyzed to determine if there were unique properties of the microorganism that contributed to efficient sugar conversion. Analysis of the genome revealed that while the Pantoea genome is similar to other Enterobacteria, such as Salmonella, Klebsiella and E. coli, in certain respects, it contains a number of different genes, which provide additional sugar metabolism capabilities.
This invention is directed to the discovery that the Pantoea citrea genome includes a family of genes that code for membrane bound three-component oxidoreductase complexes. Specifically, it was discovered that the P. citrea genome includes 19 operons that code for membrane bound three-component oxidoreductase complexes and each of these complexes include a cytochrome C homologue subunit and a subunit having dehydrogenase activity. This is in contrast to other known microbial genomes, such as Bacillus subtilis, E. coli and Pseudomonas aeruginosa. B. subtilis and E. coli are not known to include multimeric oxidoreductase enzyme complexes containing cytochrome C homologs and P. aeruginosa is known to comprise one three-component multimeric complex containing a cytochrome C homologue.